Two questions regarding in vivo cloning of mutant allele by
gap-repair in yeast.
(1) What is the best choice of cloning vector: YEp, YRp or YCp? We are
currently using YCp vector but afraid that it could disturb host genome
and the recovery efficiency is low.
(2) We are scanning a large gene in the hope to locate where the point
mutation occur in the mutant strain. Does anyone observe complicated
gap repair? e.g. the sticky ends are filled or blunted, or if two deleted
ends religate to give a deletion plasmid? These are all our "false negative"
We actually recovered the deleted plasmids when the two ends are compartible
(generated from the same restriction enzyme). How can we avoid these problems?
Department of Microbiology
University of Saskatchewan